Capillary electrophoresis (CE) is a widely used analytical and bioanalytical separation technique. It is also finding considerable use in biomedical research with new applications continually emerging. Capillary electrophoresis has been used for analysis of amino acids, peptides, proteins, nucleic acid bases, DNA oligonucleotides and numerous organic molecules. Both small ions and large biomolecules can be separated (J. Tehrani et al., High Res. Chrom., 14, 10-14 (1991)). Electrophoresis is a powerful approach for gene mapping (R. Milofsky et al., Anal. Chem., 65, 153-157 (1993)); X. Huang et al., Anal. Chem., 64, 967-972 (1992)) and DNA sequencing (H. Swerdlow et al., Anal. Chem., 63, 2835-2841 (1991)). Recently, the chemical analysis of individual cells by CE has attracted much attention (B. Hogan et al., Anal. Chem., 64, (1992); B. Hogan et al., Trends Anal. Chem., 12, 4-9 (1993)).
Polycyclic aromatic hydrocarbons (PAH) constitute a potent class of chemical carcinogens. The ability to analyze PAH in small volumes at attomole levels opens many opportunities for studying intracellular uptake, metabolism, and carcinogen-DNA adduct formation, all of which are important factors in mutagenesis and tumorigenesis. Various protocols for separation of PAH by CE have already been established. In micellar electrokinetic capillary chromatography, introduced by Terabe et al. (S. Terabe et al., Anal. Chem., 56, 111-116 (1984); S. Terabe et al., Anal. Chem., 57, 834-839 (1985); S. Terabe et al, J. Chrom., 516, 23-31 (1990)), micelles were used as a pseudophase. Nie et al. (S. Nie et al., Anal. Chem., 65, 3571-3575 (1993)) developed an approach based on solvophobic association of PAH analytes with tetraalkylammonium ions in a mixed acetonitrile-water solvent. Yan and coworkers (T. Lee, Anal. Chem., 64, 3045-3051 (1992)) demonstrated that capillary electrochromatography can be used for separation of priority PAH. Shi and Fritz (Y. Shi et al., Anal. Chem., 67, 3023-3027 (1995); Y. Shi et al, J High Res. Chrom, 117, 713 (1994)) established that excellent separation of PAH by CE can be achieved by the addition of sodium dioctyl sulfosuccinate (DOSS) to an acetonitrile-water electrolyte. Very recently, Brown et al. (R. S. Brown et al., Anal. Chem., 68, 287-292 (1996)) described a separation method for PAH using cyclodextrin-modified CE. All these approaches are able to detect PAH at sub-femtomole levels, a detectability required, for example, in the study of PAH-induced carcinogenesis.
In CE, analyte molecules are typically probed, i.e., detected, only briefly as they traverse detection zones located either on-line or in a post-column flow cell. The narrowness of this temporal detection window effectively limits the signal to noise ratio (J. Shear et al., Anal. Chem., 65, 3708-3712 (1993)). The most widely used detection method with CE is absorbance, which is usually shot-noise limited. Fluorescence, particularly laser-induced fluorescence (LIF), has also been used for detection, outperforming absorbance in sensitivity of detection by several orders of magnitude. However, the brief time available for determination of an analyte also poses a problem for LIF detection, especially when low intensity continuous wave (CW) lasers incapable of providing high induced absorption rates are used (J. Shear et al., Anal. Chem., 65, 2977-2982 (1993)). Zare and coworkers (J. Shear et al, Anal. Chem., 65, 3708-3712 (1993)) showed that velocity programming for increased detection zone residence times in CE is necessary to improve both the accuracy of quantitation and detection limits. The use of intense pulsed lasers in CE-LIF for the analysis of molecular analytes at ambient temperature produces a stronger detectable signal, but is accompanied by problem of analyte photodegradation.
Efforts to improve analyte resolution in CE are also important, particularly as detection sensitivity increases. In the case of CE-LIF, however, the emphasis has been on laser-induced fluorescence providing superior detection limits, i.e., analyte resolution is still provided by CE. At best, LIF with detection at ambient temperature can provide only very limited spectral resolution due to large vibronic fluorescence bandwidths (about 500 cm.sup.-1). Resolution between monomethylated isomers of a PAH would, for example, have to be provided by the physical separation process of CE.
It is well-recognized that analysis of chemically complex samples often requires a two-step analytical approach (separation followed by analyte characterization). Research involving analytical separation methods such as high performance liquid chromatography (HPLC) and CE has been greatly advanced by recent efforts to couple these separation techniques with sensitive spectroscopic methods that go beyond simple detection of molecular analytes by producing structural information about the separated analytes. For example, HPLC has been coupled with NMR spectroscopy for direct analysis of complex mixtures from both synthetic and biological origins. Interfacing CE with mass spectroscopy (MS) has been demonstrated; and capillary zone electrophoresis coupled with electrospray MS has been used for separation and subsequent detection of DNA adducts. Capillary electrochromatography has been coupled to MS for analysis of pharmaceutical drugs.
The utility of laser-induced fluorescence detection of molecular analytes is well-established. However, its use as a detection method in CE has been limited by the brief time available for interrogation of an analyte, and, when pulsed lasers are used at ambient temperature, photodegradation of the separated analytes. Further, laser-induced fluorescence at ambient temperature does not provide structural information about the analytes of interest. There is, therefore, a need in the art for a system that successfully interfaces fluorescence spectroscopy with a capillary electrophoresis apparatus in a manner and under conditions that allow structural characterization of closely related analytes.